Species Diversity 20: 67–71 25 May 2015 DOI: 10.12782/sd.20.1.067

Description of a New Species of Branchiobdellida (Annelida: Clitellata) and Comparison with Other Cirrodrilus Species in Northern Honshu,

Akifumi Ohtaka1,3 and Stuart R. Gelder2 1 Department of Natural Science, Faculty of Education, University, Hirosaki 036-8560, Japan E-mail: [email protected] 2 Department of Math-Science, University of Maine at Presque Isle, 181 Main Street, Presque Isle, Maine 04769-2888, U.S.A. 3 Corresponding author (Received 10 October 2014; Accepted 17 February 2015)

A new species of ectosymbiotic branchiobdellidan, Cirrodrilus iwakiensis sp. nov., is described from Cambaroides ja- ponicus (de Haan, 1849), the endemic freshwater Japanese crayfish, collected in Prefecture, northern Honshu Is- land, Japan. The anatomical features used to identifyC. iwakiensis are compared with those of its closest congener, C. nip- ponicus (Yamaguchi, 1932), as well as C. aomorensis (Yamaguchi, 1934) and C. tsugarensis Gelder and Ohtaka, 2000 distrib- uted in the same region. Key Words: Annelida, Clitellata, branchiobdellidan, taxonomy, ectosymbiont, crayfish, Japan.

dellidan. Cirrodrilus tsugarensis was described soon after the Introduction survey had finished, but specimens of the second species appeared to lack reproductive organs in segments 5 and 6. In East Asia, Branchiobdellida (Annelida: Clitellata) have Without details of the male anatomy, their generic affiliation been reported as ectosymbionts on freshwater crayfish and remained indeterminate and the species description could shrimp in China, southeastern Russia, the Korean Penin- not be completed. After 17 years of additional collecting, sula, and Japan (Ohtaka et al. 2012). Although Cirrodrilus specimens with recognizable genital organs were finally ob- is found across northern East Asia, there is no evidence to tained. This has resulted in the completion of the new spe- suggest any endemic species has been translocated between cies description presented here and also permitted an ana- the Asian mainland and Japan. Japan’s only indigenous cray- tomical comparison with other Japanese species. fish, Cambaroides japonicus (de Haan, 1849), is distributed across and northern Honshu Islands and has been reported to carry 11 species of Cirrodrilus (Yamaguchi 1934; Materials and Methods Gelder and Ohtaka 2000, 2002; Ohtaka 2010). Additional species reported from museum collections of Cambaroides Live Cambaroides japonicus were collected from 1997 japonicus include Cirrodrilus japonicus (Pierantoni, 1912) to 2014 at 21 sites on 30 occasions in (q.v.), which Yamaguchi (1935) could not authenticate and (Table 1) and taken to Hirosaki University for examina- doubted the species validity, and Branchiobdella digitata tion. Branchiobdellidans were removed from their hosts and Pierantoni, 1906 (q.v.), which Timm (1991) regarded as in- preserved in 10% formalin or 70% ethanol solutions. Speci- certae sedis. Each island has its own Cirrodrilus species, with mens were dehydrated in a graded series of ethanol and nine in Hokkaido and Cirrodrilus aomorensis (Yamaguchi, water solutions, and either prepared for mounting whole 1934) and C. tsugarensis Gelder and Ohtaka, 2000 in north- (cleared in methyl salicylate, infiltrated with Canada balsam, ern Honshu. Only one translocation is known to have oc- and slide-mounted) or for wax sectioning (8 µm thick, hae- curred between these islands, when Japanese crayfish were matoxylin-eosin-stained and slide-mounted), then exam- introduced from Hokkaido to Akita Prefecture, northern ined on a compound microscope using bright-field and dif- Honshu. These hosts carried C. inukaii (Yamaguchi, 1934) ferential interference contrast (DIC) illumination. Mounted and C. uchidai (Yamaguchi, 1932) and soon both cray- specimens have been deposited in the Zoological Institute, fish and branchiobdellidans established viable populations Faculty of Science, Hokkaido University (ZIHU), Sapporo, which have remained restricted to the translocated area Japan; the National Museum of Natural History, Smith- (Gelder and Ohtaka 2000). sonian Institution (USNM), Suitland, MD, USA, and the During a distributional study of C. japonicus in Aomori New Brunswick Museum (NBM), Saint John, NB, Canada. Prefecture from 1997 to 1999, the senior author (AO) and Anatomical terminology used in this paper follows that de- his colleagues found two undescribed species of branchiob- scribed in Gelder and Williams (2015), where “ectal” means

© 2015 The Japanese Society of Systematic Zoology 68 A. Ohtaka and S. R. Gelder nearer to the body surface, and “ental” farther from the sur- peristomial tentacles, alternating long (8) and short (7) with face. short one located at medial position, and with ventral lip medially incised and flanked by pair of small lateral lobes; Cirrodrilus iwakiensis sp. nov. 16 oral papillae present; jaws dissimilar, dorsal one larger [Japanese name: Iwaki-zarigani-mimizu] with semi-circular base narrowing to wing-like lateral tips, (Figs 1A–E, 2) large median tooth slightly curved anteriorly, and small teeth on anterior margin, ventral one with smaller, kidney- Material examined. Holotype: ZIHU 4930 removed shaped base, large median tooth and small teeth on anterior from Cambaroides japonicus collected by A. Ohtaka, 22 Sep- margin, with dental formula of 10/10 (5-1-4/5-1-4); single, tember 2012 from Oku-Shirasawa Stream on northwestern deep pharyngeal sulcus present; male organs small, glan- slope of Mt. Iwaki, Ajigasawa village, Aomori Prefecture, dular atrium club-shaped, length 0.3× segment diameter, Honshu Island, Japan (40°40′32″N, 140°15′29″E). Para- deferent lobes absent; muscular atrium tubular, length 0.2× types: ZIHU 4931, ZIHU 4932, USNM 1251839 collected segment diameter, penial sheath in ectal half, bursa small from same locality and host species as holotype by M. Mu- and spherical, penis eversible; spermatheca reduced, sper- kohyama on 16 October 1997; ZIHU 4934 from same host mathecal duct length 0.1× segment diameter, spermathecal species as holotype by A. Ohtaka, 9 September 1997 from bulb not recognized. Upper Uemata Stream, town. Additional non-type Etymology. Named after Mount Iwaki, on the slopes of specimens from same host species: ZIHU 4933 (Tokiwano, which the type locality is located. Mt. Iwaki, by A. Ohtaka, 18 June 2013), USNM 1251840, Description. Specimens 1.2–1.8 mm long, head with 1251841 (Yayoi, Mt. Iwaki, Hirosaki city, by A. Ohtaka, 11 shallow sulcus behind peristomium; body slim and pyri- May 2014), and NBM 010306–010308 (from type locality, form (Fig. 1A). Head width equal to or less than width of by A. Ohtaka and M. Mukohyama, 16 October 1997, 26 July segment 1, about equal to diameter of posterior attachment 1998, and 14 August 1998, respectively). disc. Dorsal segmental ridges, supernumerary muscles, and Diagnosis. Body slim and pyriform, 1.2–1.8 mm long dorsal or lateral appendages absent. Anterior nephridial fixed; head width equal to or less than width of segment 1; pores on segment 3 widely separated on its dorso-lateral dorsal segmental ridges, supernumerary muscles, and dorsal surface. Oral region cylindrical or funnel-shaped, surround- or lateral appendages absent; two anterior nephridial pores ed by 15 peristomial tentacles and narrow ventral lip, lat- widely separated on dorso-lateral surface; oral region cy- ter medially incised with pair of lobes flanking it (Fig. 1B). lindrical or funnel-shaped, surrounded by 15 dorso-lateral Tentacles consisting of seven short (one being median) and

Fig. 1. Cirrodrilus iwakiensis sp. nov. A, Lateral view of paratype (ZIHU 4931); B, ventral view of peristomium of paratype (ZIHU 4932), median tentacle arrowed; C, jaws (dorsal over ventral), anterior view through mouth, paratype (ZIHU 4934); D, jaws (dorsal over ventral), lateral view (m = mouth) of holotype (ZIHU 4930); E, schematic lateral view of reproductive organs in segments 5 and 6 based on holotype (ZIHU 4930) and other material, showing only their ventral half (connecting duct in segment 5 was not observed), glandular atrium dotted; muscular atrium and spermathecal duct with tangential lines; bursa and spermathecal duct with longitudinal lines). Scale bars: A, 200 µm; B, 100 µm; C, D, 20 µm; E, 50 µm. New branchiobdellidan species in Japan 69 eight long ones, alternating around dorsal and lateral mar- across its anterior margin. Ventral jaw smaller, with oval or gins of oral region (Figs 1B, 2). Oral papillae 16 in number, kidney-shaped base and both large median tooth and small surrounding mouth. Jaws dissimilar in size and shape (Fig. teeth across its anterior margin. Dental formula 10/10 (5- 1C, D). Dorsal jaw larger, having semi-circular base and ta- 1-4/5-1-4). Single, deep sulcus extending around middle of pering lateral wing-like tips, bearing large, slightly anteriorly pharynx. curved median tooth and small, conical or rounded teeth Pair of testes located in segments 5 and 6 on anterior septal wall with stages from morulae to spermatozoa free within coelom in both segments. In each segment, two prominent sperm funnels each connect to a short, vas ef- ferens which then merge into a vas deferens (Fig. 1E); both vasa deferentia enter the glandular atrium ectally. Male or- gans difficult to see, being small and often pressed against ventral body wall. Glandular atrium club-shaped, its length 0.3× segment diameter, without deferent lobes or prostate gland (Fig. 1E). Muscular atrium tubular, its length 0.2× segment diameter, with its ectal half forming a penial sheath and connected to small, spherical bursa containing eversible penis. Spermatheca with short duct, 0.1× segment diameter, passing through body wall before terminating shortly after (Fig. 1E). An anticipated ental glandular bulb has not be found to date. Variations. The peristomium may be either cylindrical Fig. 2. Cirrodrilus iwakiensis sp. nov. Frontal view of oral region or funnel-shaped, and the body is slim-pyriform to rod-like. showing arrangement of peristomial tentacles and jaws located cen- These variations reflect the worms’ reaction on contact with trally. Scale bar: 100 µm. a preservative. Dental formulae range from 10/9 (5-1-4/4-1-

Table 1. Collection records of Cirrodrilus iwakiensis sp. nov. in Aomori Prefecture, Japan, by location and date.

Latitude (N) Longitude (E) Location Date 41°07′54″ 140°31′08″ Upper Uemata Stream, Imabetsu town 09 Sep. 1997 40°59′35″ 140°35′58″ A stream in Seheji, village 22 Aug. 2006 40°55′36″ 140°30′15″ Upper Kanagi River, city 19 Dec. 1998 40°54′08″ 140°36′14″ A stream in Uchimabe, Aomori city 17 Oct. 1998 40°53′36″ 140°37′33″ Yunosawa Stream, Maeda, Aomori city 20 Aug. 2000 40°50′57″ 140°31′05″ Upper Iizume Stream, Goshogawara city 27 Aug. 1998 〃 〃 〃 09 Sep. 2000 40°50′13″ 140°33′34″ Tsuboke Stream, Mt. Manokami, Goshogawara city 31 Jul. 1998 40°49′22″ 140°31′21″ A stream in Ishidasaka, Goshogawara city 30 Oct. 2000 40°48′51″ 140°31′25″ A stream in Wakayama, Goshogawa city 27 Apr. 2000 40°47′15″ 140°31′29″ A stream in Sakaiyama, Goshogawara city 21 May 1998 40°45′18″ 140°46′06″ A stream in Yamasaki, Aomori city 30 Nov. 2011 40°44′14″ 140°43′47″ A stream in Komakino, Nozawa, Aomori city 26 Oct. 2012 〃 〃 〃 17 Nov. 2012 40°43′39″ 140°51′41″ Upper Komagome River, Aomori city 31 May 1998 〃 〃 〃 29 Jul. 2000 40°41′38″ 140°18′19″ A stream in Nagatai, Mt. Iwaki, Ajigasawa city 24 May 1998 40°40′55″ 140°15′29″ A stream in Sasamori-yama, Mt. Iwaki, Ajigasawa village 16 Oct. 1997 40°40′50″ 140°15′47″ Oku-shirasawa Stream, Mt. Iwaki, Ajigasawa village 16 Oct. 1997 〃 〃 〃 26 Jul. 1998 〃 〃 〃 14 Aug. 1998 〃 〃 〃 22 Sep. 2012 〃 〃 〃 12 Oct. 2012 40°40′52″ 140°40′42″ A stream in Hosono, Aomori city 25 Jun. 2000 40°40′17″ 140°21′00″ Kosugisawa Stream, Mt. Iwaki, Hirosaki city 11 May 2014 40°39′16″ 140°21′23″ A stream in Yayoi, Mt. Iwaki, Hiorosaki city 11 May 2014 40°37′08″ 140°15′41″ A stream in Tokiwano, Mt. Iwaki, Hirosaki city 13 Nov. 1997 〃 〃 〃 07 Jul. 1998 〃 〃 〃 18 Jun. 2013 40°37′08″ 140°15′41″ A stream in Moriyama, Mt. Iwaki, Hirosaki city 11 Aug. 2005 70 A. Ohtaka and S. R. Gelder

4) to 11/11 (5-1-5/5-1-5) and maybe greater; however, the by AO resulted in discovery of a restricted population of respective jaw base shapes and sizes are all alike. The small, Cirrodrilus tsugarensis (see Gelder and Ohtaka 2000) as well short spermatheca is rarely recognizable as it is pressed as the more widely distributed Cirrodrilus iwakiensis (Table against the ventral body wall. 1). Describing C. iwakiensis for publication was delayed Distribution and habitat. The new species is found in until specimens with recognizable male reproductive organs the western half of Aomori Prefecture in northern Honshu in segments 5 and 6 could be found. At first it was assumed (Table 1). that the specimens were immature because their male ducts Japanese crayfish were collected from small, cold, head- were either very small or appeared absent; however, the water streams with small rocks interspersed with patches of presence of mature spermatozoa and sperm funnels negated gravel and thin branches. Specimens of C. iwakiensis were this explanation. Finally, two specimens with recognizable not found in the branchial chamber, but on their hosts’ cara- male genitalia were found and their anatomy confirmed pace, abdomen, and appendages, among which no preferen- them to be members of Cirrodrilus. Without this confirma- tial site can be discerned. tory evidence, the species might have been placed in the Host. Cambaroides japonicus. wrong genus, resulting in it being considered a species inqui- Remarks. Specimens of Cirrodrilus iwakiensis always renda. The vas deferens in segment 5 was not observed (Fig. occurred with more numerous C. aomorensis on a host. 1E), although given the sperm funnels and vasa efferentia Specimens were collected throughout the year except dur- its presence is predicted. No complete spermatheca was ing January to March, when snow and ice prevented ac- observed in any of the 74 specimens examined during the cess to the collection sites. Examination of gut contents of preparation of this description, only a spermathecal duct as C. iwakiensis showed no appreciable change in composition it passed from the exterior into the ventral segment coelom. over the seasons. The diet consisted of brown, amorphous The presence of a spermathecal bulb is likely, but remains to flocculent masses with diatom frustules, algal debris (cell be confirmed; however, this does not negate the validity of walls), and mineral particles in small amounts. Occasionally, the species. This situation is not unique in branchiobdelli- small bodies containing nucleated cells and larger unidenti- dans, as a spermathecal bulb in Cambarincola speocirolanae fiable fragments, also nucleated, were observed. Holt, 1984 (q.v.) was not observed but hypothesized. The new species is clearly distinguishable from its conge- ners by its peristomial tentacle arrangement and jaw struc- Discussion ture (cf. Timm 1991: fig. 46) as well as by its having much smaller male organs. Cirrodrilus iwakiensis most closely re- Until the 1990s, only one species of branchiobdellidan, sembles the Hokkaido species Cirrodrilus nipponicus (Yama- Cirrodrilus aomorensis, was known to be indigenous to Ao- guchi, 1932), which also has a funnel-shaped peristomium mori Prefecture, northern Honshu. Extensive collecting of with eight long and seven short tentacles arranged alternate- the Japanese crayfish, Cambaroides japonicus, and more im- ly. However, the jaws in C. nipponicus are similar in size and portantly a detailed examination of their branchiobdellidans the lateral tips curl medially (Yamaguchi 1932), in contrast

Fig. 3. A–C, Cirrodrilus aomorensis (Yamaguchi, 1934); D–F, C. tsugarensis Gelder and Ohtaka, 2000. A, D, Ventral view of peristomium showing tentacles (median one arrowed) and ventral lip; B, E, jaws (dorsal over ventral), anterior view through mouth; C, F, jaws (dorsal over ventral), lateral view (mouth=m). Scale bars: A, D, 100 µm; B, C, 10 µm; E, F, 20 µm. New branchiobdellidan species in Japan 71 to the distinctly larger dorsal jaw with wing-like lateral tips structive comments on the manuscript. in C. iwakiensis. In addition, the dental formula in C. nip- ponicus is 15/13 (4-1-7/6-1-6), the teeth being more numer- ous than in C. iwakiensis. References In view of the recently recognized presence of Cirrodri- lus aomorensis, C. tsugarensis, and C. iwakiensis in northern Gelder, S. R. and Ohtaka, A. 2000. Description of a new species and a Honshu, their defining anatomical characteristics need to redescription of Cirrodrilus aomorensis (Yamaguchi, 1934) with a be examined. The most obvious external character is that C. detailed distribution of the branchiobdellidans (Annelida: Clitel- lata) in northern Honshu, Japan. Proceedings of the Biological So- iwakiensis has 15 tentacles whereas the others have 13. All ciety of Washington 113: 633–643. of the tentacles of C. tsugarensis are of the same length (Fig. Gelder, S. R. and Ohtaka, A. 2002. A review of the oriental branchiob- 3D), intermediate in length between the long and short ten- dellidans (Annelida: Clitellata) with reference to the rediscovered tacles of C. iwakiensis (Figs 1B, 2) and C. aomorensis (Fig. slide collection of Prof. Hideji Yamaguchi. Species Diversity 7: 3A). The latter two species both have a short median tenta- 333–344. cle, but C. aomorensis has two pairs of long tentacles on the Gelder, S. R. and Williams, B. W. 2015. Clitellata: Branchiobdellida. Pp. dorsal lip while C. iwakiensis has alternating long and short 551–563. In: Thorp, J. and Rogers D. C. (Eds) Ecology and Biology: tentacles around the margin of the ventral lip. The dorsal Thorp and Covich’s Freshwater Invertebrates. Academic Press, New jaw in C. iwakiensis (Fig. 1C) and C. aomorensis (Fig. 3B) York. Holt, P. C. 1984. On some branchiobdellids (Annelida: Clitellata) from is larger than the ventral jaw, but in C. tsugarensis they are Mexico with the descriptions of new species of the genera Cam- similarly sized (Fig. 3E). Both species with larger dorsal jaws barincola and Oedipodrilus. Proceedings of the Biological Society have a large median tooth that curves anteriorly towards the of Washington 97: 34–42. mouth (Figs 1D, 3C), in contrast to the straight tooth in C. Ohtaka, A. 2010. [Community conservation]. Pp. 445–475. In: Kawai, tsugarensis’ (Fig. 3F). The dorsal jaw in C. iwakiensis is semi- T. and Takahata, M. (Eds) Biology of Crayfish. Hokkaido Daigaku circular with lateral curved “wings” (Fig. 1C), but the latter Shuppankai, Sapporo. [In Japanese] are absent in C. aomorensis (Fig. 3B). Although each species Ohtaka, A., Gelder, S. R., Nishino, M., Ikeda, M., Toyama, H., Cui Y.-D., has a different dental formula, natural variation from the He, X.-B., Wang, H.-Z., Chen, R.-B., and Wang, Z.-Y. 2012. Distri- worm may add or subtract a small tooth, so this character butions of two ectosymbionts, branchiobdellidans (Annelida: Cli- should only be used with caution. tellata) and scutariellids (Platyhelminthes: “Turbellaria”: Temno- cephalida), on atyid shrimp (Arthropoda: Crustacea) in southeast Our recent survey of Japanese crayfish and their bran- China. Journal of Natural History 46: 1547–1556. chiobdellidans in Aomori Prefecture has resulted in two Pierantoni, U. 1906. Nuovi Discodrilidi del Giappone e della California. new species of ectosymbionts being described. No new spe- Annuario del Museo Zoologico della R. Università di Napoli (New cies of Cirrodrilus have been reported in Hokkaido since Series) 2: 1–9. the work of Yamaguchi (1934). Given the present results, it Pierantoni, U. 1912. Monografia dei Discodrilidae. Annuario del Museo is probable that new species will be found if a comparable Zoologico della R. Università di Napoli (New Series) 3: 1–27. crayfish and branchiobdellidan survey is conducted there. Timm, T. 1991. Branchiobdellida (Oligochaeta) from the farthest South-East of the U.S.S.R. Zoologica Scripta 20: 321–331. Yamaguchi, H. 1932. Description of a branchiobdellid, Carcinodrilus Acknowledgments nipponicus n. g. et n. sp. Journal of the Faculty of Science, Hok- kaido University, Series VI, Zoology 2: 61–67. Yamaguchi, H. 1934. Studies on Japanese Branchiobdellidae with some We thank Takaki Naraoka, Masaaki Oikawa, Koji Sa- revisions on the classification. Journal of the Faculty of Science, samori, Nariyuki Sato, and the late Mitsuru Mukohyama for Hokkaido University, Series VI, Zoology 3: 177–219. providing specimens used in the present study. In addition, Yamaguchi, H. 1935. Family Branchiobdellidae (Class Clitellata)]. Pp. our appreciation goes to Tarmo Timm, Bronwyn W. Wil- 1–37. In: Okada, Y., Uchida, T., and Ezaki, T. (Eds) Fauna Nipponi- liams, Mark J. Grygier and Hiroshi Kajihara for their con- ca, Vol. 6, Fasc. 3, No. 2. Sanseido, Tokyo. [In Japanese]